1. Field of the Invention
This invention is directed to compositions of matter and methods employing one or more of these compositions. More specifically, this invention concerns a novel lytic reagent system for rapidly effecting hemolysis of the erythrocyte fraction of whole blood and thereby permitting the isolation of the leukocyte fraction in its native or near native state. The leukocyte fraction may then be subjected to further study or analysis in a variety of environments. One of the preferred uses of this lytic reagent system is the pretreatment of the whole blood sample to effect rapid and essentially complete hemolysis of the erythrocyte fraction. Such pretreatment also results in subtle modification to the leukocyte fraction, thus facilitating its further differentiation into at least five (5) distinct sub-populations.
2. Description of the Prior Art
The separation of complex biological fluids (i.e. whole blood) into its various constituents preliminary to study and analysis of its component parts, is generally desirable and often an essential requirement of many established analytical protocols and/or instrumentation utilized in such studies and analysis. Where such study or analysis of the fluid fraction of the sample is of primary interest, the cellular fraction is separated from the sample without regard for maintaining the cell viability or membrane integrity. Conversely, where the cellular fraction is itself of primary interest to the researcher or clinician, the partitioning of the whole blood sample into its various cellular components requires that the sample treatment/processing techniques be adjusted accordingly. The traditional methods for separation of whole blood samples into its various cellular components is by centrifugation. While this process is effective, it is labor intensive, relatively inefficient and requires physical manipulation of the cellular fraction of the sample.
Where such separation/partitioning of the cellular fraction of the sample is attempted with chemical agents, the results have been less than totally satisfactory for a variety of reasons. More specifically, the vitality and viability of a cell population in vivo or in vitro is dependent upon maintaining a precise physiological environment that is consistent with the preservation of physical cell structure and chemical balance within the cell. This balance is controlled by the permeability and transport characteristics of the cell membrane. Alteration in the physiological environment will in turn evoke a response or change in the cell membrane. The membrane response is "defensive" in nature; that is, the physiological response of the membrane is calculated to maintain the chemical balance within the cell and, thus, its continued and uninterrupted vitality.
It is fully appreciated that such alteration in the ideal physiological environment of the cell can be tolerated only within limits; and, that when such limits are exceeded, permanent injury to the cell can occur. As is further appreciated in the art, such changes in environment (i.e. tonicity of the diluting medium--even with distilled water) can effect hemolysis of the cells.
The degree of tolerance of various cell populations in whole blood to changes in their physiological environment has been extensively studied and documented. The effects of alteration in various aspects of the physiological environment of the cellular fraction of whole blood are both subtle and dramatic and can be effected through dietary metabolites and/or foreign substances. These studies include monitoring the reaction of cellular preparations to different drugs, Da Costa, A. J. et al, Transfusion, (1973), 13, 305; to dietary imbalance, Kobayashi, T. et al, Journal of Biochemistry (1983) 93, 675; and to changes in pH, Rother, U. et al, Z. Immunologie Forschungsgemeinschaft (1978) 155, 118, and Schettini, F. et al, Acta Paediat. Scand. (1971), 60, 17.
In each of the articles noted above, a drug, food metabolite or change in pH resulted in significant alteration in the physiological environment of the blood to the degree where hemolysis of the red blood cells was effected.
More specifically, the above referenced Rother article reports serum activation by acidification (pH 6.4) with hydrochloric acid which lysed unsensitized erythrocytes in the presence of EDTA. The article compares the effect of such acidification with the "deviated lysis" activity observed following serum activation with insulin. An independent and unrelated study by Schettini and his co-workers concluded that red blood cells from infants and young children were more sensitive to acid hemolysis than red blood cells from older individuals.
As of the present, no chemical treatment is presently available to rapidly and effectively partition a whole blood sample into viable cellular fractions. Where one or more chemical treatments of the whole blood sample is used (as in the preparation or pretreatment of whole blood for the performance of white blood cell differentials), the focus of such treatment has been to alter the sample to permit the differentiation of its cellular components from one another based upon isolation/analysis of (i) the cellular debris (i.e. nuclei) remaining subsequent to such treatment; (ii) the fixation of the cells by such chemical treatments; or (iii) the relatively severe modification of such cells which eventually results in their ultimate disintegration. Such relatively harsh and disruptive chemical treatments of whole blood samples have, however, been successfully applied where combined with relatively sophisticated instrumentation. More specifically, the ability to alter the physiological environment of a cellular population in vitro has been used to advantage in the measurement of certain cellular parameters and to quantitate the individual populations. The distinctive reaction of each individual cell population to a change in its physiological environment has particular advantages in the identification of the individual leukocyte sub-populations of cells of whole blood. The leukocyte population of blood has been classified previously into two major fractions: the lymphoid and the myeloid fraction. The lymphoid fraction consists of lymphocytes (B and T cells). The myeloid fraction consists of monocytes and granulocytes (neutrophils, basophils and eosinophils). One accepted technique for modification of the physiological environment of the cellular population of whole blood has been through the addition of certain so-called "lytic reagents" to a blood sample. The development of certain lytic reagents and lytic reagent systems has provided the clinician with the ability to effectively isolate the white cell population (hereinafter "leukocytes") from the red cell population of whole blood. The relative concentration of leukocytes within the blood sample and the gross morphological appearance of certain classes of these cells can be clinically significant.
This ability to further differentiate the leukocyte population, thus, provides an invaluable diagnostic tool in the study and treatment of various diseases. As is further appreciated, the larger the number of sub-populations of leukocytes which are identifiable, the more accurate and reliable the identification of any one such sub-population.
A number of references have appeared in the recent patent literature which disclose various reagent systems and techniques for enhancing the ability of automated instrumentation to conduct white blood cell differentials. The following references are representative of the pertinent patent literature in this field: U.S. Pat. Nos. 3,874,852; 4,286,963; 4,346,018; 4,485,175; 4,520,274; 4,529,704; and U.S. applications Ser. No. 615,961 (corresponding International Application PCT/US85/00954, published Dec. 19, 1985); and, Ser. No. 615,966 (corresponding International Application PCT/US85/00868, published Dec. 19, 1985), all of which are hereby incorporated by reference in their entirety.
U.S. Pat. No. 3,874,852 (to Hamill) describes a reagent system and method useful in the performance of leukocyte and hemoglobin determinations of whole blood. This reagent system comprises an essentially ferrocyanide free aqueous solution of quaternary ammonium salt and cyanide ions. This reagent system is effective to stromatolyze both red blood cells and platelet cells in whole blood and in the conversion of the free hemoglobin to a chromogen. This system is reported efficacious for leukocyte and hemoglobin determinations with diagnostic accuracy. The leukocyte population profile available with this system is, however, limited to total white cell count, without further differentiation of this cellular fraction into its discrete sub-populations.
U.S. Pat. No. 4,286,963 (to Ledis, et al) describes a lytic diluent and method for achieving rapid lysis of red blood cells in whole blood. This diluent enhances the ability of automated instrumentation to perform differential determinations of lymphoid and myeloid sub-populations of leukocytes and the quantitative determination of hemoglobin. The lytic diluent described by Ledis is composed of a mixture of at least one quaternary ammonium salt and an aryl substituted short chain alkanol in buffered aqueous medium (pH 3.5 to 5.0). The lytic diluent of this Ledis patent is, however, limited in its ability to effect differentiation of the leukocyte population into the two (2) principle sub-populations; namely, the lymphoid and myeloid fractions.
U.S. Pat. No. 4,346,018 (to Carter, et al) describes a multipurpose blood diluent and a method for utilizing this diluent in combination with a weak lysing reagent system for the performance of hemoglobin determination and the differentiation of lymphocytes into the lymphoid and myeloid sub-populations. This diluent comprises, among other constituents, N-(2-acetamido)iminodiacetic acid (ADA) as a blood stabilizing agent. The lysing agent comprises an aqueous solution of at least one quaternary ammonium salt. The diluent/lytic reagent of this Carter patent is, however, limited in its ability to effect differentiation of the leukocyte population into the two (2) principle sub-populations; namely, the lymphoid and the myeloid fractions. In addition, ADA has been found to help stabilize the size distribution, cellular shape, and most importantly, the high degree of cellular dispersion of erythrocytes and platelets to an extent not previously observed with other compounds.
U.S. Pat. No. 4,485,175 (to Ledis, et al) describes a reagent system and method for performance of differential determinations of leukocytes into three (3) sub-populations utilizing automated cell counting equipment. This reagent system comprises a blood diluent and a lysing agent. The lysing agent (comprising an aqueous mixture of quaternary ammonium salts), when added to the diluted blood sample under mild conditions of concentration and at a relatively slow rate, causes unexpected volume modifications to the various leukocyte sub-populations. The discovery which permitted the attainment of the degree of differentiation of the leukocyte population by Ledis, et al is based upon the observation of the relative greater sensitivity of the granulocyte sub-population to lytic agents. By controlling the rate of exposure of the lymphocyte population to lytic agents, the granulocyte sub-population is better preserved. The reagent system of this Ledis et al patent is, however, limited in its ability to effect differentiation of the leukocyte population into three (3) sub-populations; namely, lymphocytes, monocytes and granulocytes.
While all of the above lytic agents and reagent systems facilitate the differentiation of the leukocyte fraction of a blood sample (to a greater or lesser degree), each suffers from a common deficiency; namely, the inability to effect such differentiation without adversely altering the chemical balance of the cells which are subjected to such treatment. Where such alteration in the chemical balance is induced, the effect on the cellular population can range from relatively minor changes (i.e. swelling) to lysis. Dramatic chemical changes in the physiological environment of the leukocyte population also alters the immunochemical response of the leukocyte surface markers. The treatment of leukocytes with such traditional lytic agent system is, thus, inherently incompatible with further immunochemical study of these leukocytes. This limitation has thus, up to now, prevented the use of lytic reagents, alone or in combination with other means, for further refinement in the diagnostic process of various disease states, based upon the differences in the immunochemical response of the respective surface markers of each such cell population.